This proposal outlines a plan to investigate the assembly mechanism of the 30S ribosomal subunit, specifically to determine the pathways of assembly in vitro and to measure the effect of co-transcriptional assembly. Our aim is to answer questions about the extent of cooperativity in the assembly process and the nature of the rate-limiting step(s) by (1) measuring binding rates of individual ribosomal proteins and detecting any intermediates that are populated during assembly by three-color, two-photon excitation fluorescence and (2) designing an in vitro co-transcriptional assay for 30S assembly. Method: In order to obtain kinetic information on possible intermediates, we are constructing a two-photon excitation three-color detection fluorescence microscope that allows us to measure kinetic rates at the single molecule to small ensemble level. To measure the fluxes in real-time we will use time dependent fluorescence correlation spectroscopy (PCS)to study the relationships of up to three proteins at a time, each labeled with a different fluorescent tag. We will measure the relative rate constants and look for kinetic cooperativity by monitoring if the rates of a particular protein change in the presence of another protein. By auto-correlating the fluorescence intensity signal we will determine the concentrations of each species in time and individual binding rates and quantify higher order molecular complex formation using cross-correlation measurements. Relevence: A properly functioning ribosome is crucial to cell viability and understanding how the assembly process is regulated has a multitude of implications in biology. These studies will help to quantitatively define the energy landscape of 30S and yield mechanistic insights into the assembly process by identifying 1) if multiple pathways can lead to the formation of the intact active ribosome and what these pathways are 2) the composition of any intermediates and 3) the effect of co-transcriptional assembly on the rate of the assembly process.